Thrombin is a highly specific enzyme catalyzing the proteolytic cleavage of fibrinogen to fibrin in blood congulation. Thrombin is also involved in cleavage of a 37 residue amino acid peptide from Factor XIII which, upon activation, catalyzes formation of the intramolecular gamma-glutamyl-lysine bridges cross-linking fibrin molecules. Thrombin is generated from its precursor prothombin, and several molecular forms (depending on the extent of cleavage) have been identified. If available, therapeutically purified human thrombin would have many beneficial applications. For example, human thrombin can be effectively used as a topical agent to promote coagulation. It can be incorporated into a polymer structure for use as a wound dressing. A still further application is in compositions known as fibrin glue in which thrombin and fibrin contained in separate capsules are combined for immediate application to fresh wounds, particularly surgical incisions.
Bovine thrombin was first purified in analytical quantities by Rasmussen utilizing an ion exchange resin IRC-50 (P. S. Rasmussen, Biochem. Biophys. Acta, 16, 157 (1955). Strassle, et al., Biochem. Biophys. Acta, 73, 462 (1983) purified thrombin on DEAE-cellulose to obtain a preparation of high specific activity (2100 NIH units/mg), and also discovered that such high specific activity could be attained if the thrombin was first produced from its zymogen.
Conversion of prothrombin to thrombin by proteolysis is dramatically increased in the presence of a phospholipid bilayer, calcium ions, and Factor V. For a general review of the biochemistry of thrombin formation, see Bloom & Thomas, Hemostasis and Thrombosis, 2 ed., (Churchill Livingstone, 1987). Accordingly, purification schemes in which the thrombin is derived from proteolytic conversion of prothrombin must include factors promoting such conversion. Alternatively, the lipoprotein thromboplastin may be utilized to activate Factor VII which in turn catalyzes formation of the Factor Xa mediating conversion of prothrombin to thrombin.
Other purification techniques are known in the art. Thompson & Davie, Biochem, Biophys. Acta, 250, 210 (1971) purified thrombin by affinity chromotography on p-chlorobenzyl amino-e-aminocaproyl agarose. Schmer, Hoppe-Sejler's, Z. Physiol. Chem., 353, 810 (1972) utilized a combination of ion exchange and affinity chromatography. All of these methods involve purification of thrombin from bovine plasma, which is a readily available and inexpensive source for analytical study.
There are, of course, serious limitations to the use of bovine thrombin in human therapeutic applications. First, the impurities present in bovine preparations include bovine proteins which are antigenic. Secondly, bovine thrombin itself, like other proteins of animal origin for which a homologous human species exists, may cause anaphylactic reactions, even when applied topically (see Thrombosis Research. 53, 277 (1989). The lack of a high purity preparation of human thrombin has frustrated attempts to use thrombin as a local hemostat in eye surgery (see Aaberg, Am. J. Opthomology, 106, 485 (1988), because of adverse reactions.
In the field of the purification of human thrombin, which differs molecularly from the bovine equivalent, less research has been done. At present there are two basic approaches to purification. One approach was developed by Fenton, et al., Chemistry and Physiology of Human Plasma, ed. D. H. Bing, Pergammon Press: NY (1979), utilizing polymethyl acrylic acid which reportedly has a quantitative adsorption affinity for thrombins and can be used to selectively separate the beta and gamma species of thrombin from the alpha species. As noted by this investigator, human thrombin obtained in this procedure tends to be unstable and very prone to autolytic degradation.
The second basic approach utilizes a sulfated polysaccaride matrix to selectively sorb thrombin, Lundblad, Biochemistry, 10, 2501 (1971). While this matrix yields a thrombin product of high specific activity, the use of the sulfated Sephadex.RTM. matrix is unsuitable for production of therapeutic-grade thrombin. The Sephadex.RTM. derivative is a fine powder with the adverse characteristic of wide fluctuations in swelling and shrinking under various solvent conditions. It is also very difficult to prevent contaminating powder fines from being introduced into the final product.
Other purification methods have been reported more recently. Mullen and Yu, J. Chromatography, 359, 351 (1986) describe a method of purifying thrombin on cross-linked polystyrenes modified with L-arginyl methylester which mimics the emzymatic binding of thrombin substrates. The method is particularly advantageous for analysis of very small amounts of thrombin prone to degradation. An improved purification technique was reported by Nordenman et al., Thrombosis Research, 11, 799 (1979) involving affinity chromatography on heparin immobilized onto agarose. The thrombin product appeared to be of greater purity upon electrophoresis than that obtained by some previous methods, but there was no indication that this technique or that described by Muller and Yu can be adapted to large-scale production.